Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid‐induced DNA damage

Abstract Background The risk of esophageal adenocarcinoma (EAC) is associated with gastro‐esophageal reflux disease (GERD) and obesity. Lipid metabolism‐targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. Methods Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO‐1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. Results Metabolism‐focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro‐resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE‐BE‐EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4‐Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6‐Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post‐translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid‐induced DNA double‐strand breaks in FLO‐1 cells in vitro. Conclusions Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid‐induced DNA damage in vitro, potentially through reduced lipid peroxidation.

. List of lipid abbreviations P2 Table S2. Lipidomics data see separate excel sheet Table S3. Patient cohort information for proteomics/lipidomics and transcriptomics cohorts. P3 Table S4. Joint loadings from JIVE analyses see separate excel sheet   Table S4. Joint loadings from JIVE analyses see separate excel sheet Figure S1. Sample exclusion process. PCA score plots of patient biopsy (A) lipidomics data (n = 586 lipids) and (B) proteomics data (n = 3387 proteins). Dot color indicates the sample disease phenotype. Data was autoscaled prior to model generation. Ellipse: Hotelling's T2 (95%). Log 2 abundance of protein markers for intestinal metaplasia, analysed using MS proteomic techniques on biopsy tissue. Two BE samples (BE6 and BE7) clustered with normal squamous epithelium in both lipid and protein PCA plots. When analysed for protein abundance for intestinal metaplasia markers (C), these samples resembled squamous epithelium, and were excluded from further analyses.     The source capillary voltages were set to 3500 V and auto-recalibration using known reference masses (69.10, 112.99 and 1033.99 Da) was enabled. The reversed phase buffers used for negative ionization mode were the same as used in positive ionization, except for the addition of 10 mM ammonium acetate instead of the previously used ammonium formate and formic acid.

Lipidomics: patient samples
Lipids with a coefficient of variation (CV) > 30% among the quality control samples were removed from the dataset. For the untargeted lipidomics data, linear models of HPLC retention times were produced for the lipid classes PC, PE, LPC, LPE, PI, Cer, SM and TG. These models allowed the assessment of expected and measured retention times based on the lipid class, chain length and number of unsaturated bonds. Additionally, Kendrick mass defects (KMD) were calculated to ensure that the referenced KMD (RKMD) were consistent with the number of unsaturated bonds in the identified lipid molecule. Lipids for which the RKMD were not in agreement with the number of unsaturated bonds were excluded from the analyses.

Lipidomics: cell samples
Lipid extraction: Briefly, cell pellets were resuspended in 10μL of ice-cold milli-Q water before addition of 200μL ice-cold butanol/methanol (1:1) containing 50μg/mL antioxidant butylated hydroxytoluene (BHT) and 10mM ammonium formate. After vortexing, 10μL of a 1/10-diluted internal standard lipid mixture (SPLASH Lipidomix, Avanti #330707) was spiked into each sample to assess sample preparation and liquid chromatography retention time consistency across samples. The samples were incubated in a thermomixer for 1 hour at 4°C/850rpm before centrifugation for 15 minutes at 4°C/21,000g. The supernatant was removed and dried down in a vacuum concentrator for approx. 120 minutes. The resultant residue was resuspended in 50μL methanol/toluene (9:1) containing 100ng/mL 1-cyclohexyl-dodecanoic acid urea (CUDA). CUDA serves as an internal standard to monitor autosampler injection inconsistencies. Finally, samples were centrifuged for 5 minutes at 4°C/21,000g and the supernatant was transferred to a vial for analysis by LC/MS. In addition, a 5 L aliquot of each sample was combined to prepare a pooled QC sample to aid preliminary spectra analysis.

Proteomics
Protein pellets were resuspended in 45 µL lysis buffer containing 10 mM TCEP, 40 mM 2CAA and 1% SDC in 100 mM TRIS. Samples were heated at 95°C for 5 min, followed disruption in a Bioruptor ultrasonicator (Diagenode, Liege, Belgium) (10 cycles of 30 sec vortex, 30 sec rest). After centrifugation for 10 min at 5000 × g, the supernatant was collected as tissue lysate. Protein concentration was determined by spotting 2 µL lysate on Millipore DirectDetect cards (Merck, Sydney, Australia) and dried before measurements. Lysis buffer was used as a blank, measurements were made using the AM2.q3 setting. Trypsin digestion was performed at 37°C with 20 µg sample in 24 µL lysis buffer, and 0.4 µg sequencing grade modified trypsin (Promega #V5111) in 180 µL trypsin resuspension buffer. After overnight incubation, samples were acidified using 10 µL of 10% TFA and centrifuged at 3,200 × g for 10 min to remove particulates. The supernatant (160 µL) was collected and cleaned using Glygen 250 µL C-18 Velotips (Velo-C18-20µg). Solvents were removed by drying in a Savant Speedvac concentrator (Thermo Fisher Scientific, Brisbane, Australia) at 45°C. Dried peptides were stored at -20°C until use, then resuspended in 0.1% formic acid in MilliQ water prior to mass spectrometry analysis.
Trypsin digested samples were separated using the Thermo Easy-nLC 1000 with NanoViper separation column (#6041.5261) and Acclaim PepMap 100 NanoViper C18 trap column (#164705). A 180-minute gradient of buffer A (0.1% formic acid in MilliQ water) and buffer B (0.1% formic acid in acetonitrile) was run with a 180min gradient (Flow 250nl/min, Time 0min: 3%B, 3min: 6%B, 143min: 25%B, 163min: 40min, 164min 95%B, 179min 95%B, 180min: 3%B). Peptides were analyzed on a Thermo Orbitrap QE plus mass spectrometer. The Chromatography Peak width was set to 12 seconds. Dynamic exclusion was set to 10 ppm. A full MS / dd-MS2 (TopN) was run in positive polarity with an in-source collision-induced dissociation of 0 V. Default charge state was set to 2, microscans set to 1 and a resolution of 70,000 was used. The acquisition target was set to 3e6 with a maximum injection time of 100 ms. The scan range was 350 to 1400 m/z and the spectrum "profile" data type was used. The dd-MS2 / dd-SIM settings were set to a resolution of 17,500, automatic gain control target of 5e5, maximum injection time of 55 ms, loop count of 20, MSX count of 1 and TopN of 20. The isolation window was set to 1.2 m/z, isolation offset of 0 m/z, scan range from 200-2000 m/z and fixed first mass of 140 m/z. NCE was 29, spectrum data were saved as centroid. MaxQuant version 1.6.1.0 was used for feature extraction and protein identification with the human Swiss-Prot database containing 20,258 reviewed proteins (downloaded February 2018). The digestion enzyme was set to Trypsin/P with a maximum of two missed cleavages and carbamidomethyl as fixed modification. Variable modifications were set to oxidation and acetylation with a maximum number of five variable modifications per peptide. Default instrument settings for Orbitrap mass spectrometers were used. Match type was set to "Match from and to". Sequence data from Swiss-Prot was loaded and carbamidomethyl fixed modifications were allowed. Minimum peptide length was set to 5 and maximum mass to 4600 Da, peptide length for unspecified search was set between 6 and 25. For identification a score of 30 was required for modified peptides and at least one razor or unique peptide needs to be detected. The "match between runs" parameter was set to a match time window of 4 minutes and an alignment window of 25 minutes to recursively identify features between samples. For quantitation the label minimum ratio count was set to two, using only unique and razor peptides (modified and unmodified) with advanced ratio estimation. Large LFQ ratios were stabilized and advanced site intensities was selected.

Immunohistochemical staining
Sections were first affixed to positively-charged adhesive slides and air-dried overnight at 37°C before dewaxing and dehydration with descending graded alcohols and water. Sections were then incubated in Tris Buffered Saline (TBS) at pH 7.6 with 2% (FADS2) or 3% (FADS1, Nrf2) H2O2 for 10 minutes to block endogenous peroxidase activity. Sections were washed in three changes of water and transferred to Dako Epitrope Retrieval Buffer (FADS2: pH 9.0, 20min at 100 o C heat retrieval. FADS1: pH6.0, 20min at 80 o C. Nrf2: pH 6.0, 5min at 125 o C). After cooling for 20 minutes, the sections were washed in TBS with 0.05% Tween (TBSTW). Biocare Medical Background Sniper and 2% BSA were added for 15 minutes to inhibit non-specific antibody binding. Excess Sniper BSA was removed from the sections. FADS2 primary antibody (Abcam#ab232898) was diluted 1:25 in Da Vinci Green antibody diluent and applied overnight at room temperature. FADS1 antibody (Abcam#ab236672) was diluted 1:200, DEGS1 antibody (Abcam#ab167169) was diluted 1:200, and Nrf2 antibody (Abcam#ab137550) was diluted 1:700 in Da vinci Green and applied one hour at room temperature. Sections were washed three times in TBSTW and Biocare Medical MACH1 Universal was applied for 20 minutes (30minutes for Nrf2). The sections were again washed in TBSTW before signals were developed in NovaRed for 3 minutes, or DAB for 2 minutes (Nrf2). To remove excess chromogen, the sections were washed in water before the application of a light haematoxylin